The mass spectrometry imaging method is a technique of examining a distribution of a substance having a specific mass by performing mass spectrometry on a plurality of measurement points (minute regions) in a two-dimensional region of a sample such as a biological tissue piece. Applications for drug discovery, biomarker exploration, and investigation of cause of various sicknesses and diseases are being studied in the mass spectrometry imaging method. A mass spectrometer that performs the mass spectrometry imaging method is generally called an imaging mass spectrometer (see Patent Documents 1 and 2 and Non-Patent Document 1). Usually, microscopic observation is performed first on some two-dimensional regions on the sample, and then an analysis target region is decided based on the microscopic observation image and the imaging mass spectrometry of the region is performed. Consequently, the imaging mass spectrometer is called a microscopic mass spectrometer or a mass microscope, and referred to as “imaging mass spectrometer” in the present description.
In the imaging mass spectrometer, an ion source in which a matrix assisted laser desorption ionization (MALDI) method is adopted is normally used. In the ion source in which the MALDI method is adopted, a surface of the sample is irradiated with a laser beam whose diameter is narrowed by a condensing optical system including a lens, and ions of the substance contained in the sample are generated at and around the region of the laser beam irradiation. The generated ions are extracted from the surface of the sample by action of an electric field, introduced to a mass spectrometer through an ion transport optical system or the like as needed, separated according to a mass-to-charge ratio, and detected. The substance in the sample is ionized in an atmospheric pressure atmosphere or a vacuum atmosphere.
In a typical imaging mass spectrometer, in producing a mass spectrometry image in a two-dimensional analysis target region having a predetermined shape on the sample, the mass spectrometry is repeated by irradiating the sample with the laser beam in a pulsed manner while a sample stage on which the sample is placed is moved with a predetermined step width in two orthogonal axes (X-axis, Y-axis) in the two-dimensional plane. At this time, the laser beam irradiation region on the sample surface normally has a substantially circular shape or a substantially elliptical shape. On the other hand, each pixel (pixel) on the mass spectrometry image produced based on a mass spectrometry result has a rectangular shape. For this reason, it is necessary to associate substantially circular or substantially elliptical laser beam irradiation region with the rectangular pixels on the mass spectrometric image.
FIGS. 8(a) to 8(e) are schematic diagrams illustrating an example of the association between substantially circular laser beam irradiation region (the minute region where the mass spectrometry is actually performed) and the rectangular pixel on the mass spectrometry image. As illustrated in FIG. 8(a), it is considered that the mass spectrometry is performed on each of rectangular unit attention regions 102 obtained by dividing a two-dimensional (in an X-Y plane) analysis target region 101 set on a sample 100 into a lattice shape. One unit attention region 102 corresponds to one pixel on the mass spectrometry image. Though, in practice, the analysis target region 101 need not have a rectangular shape, it is assumed here that the analysis target region 101 is also rectangular for the purpose of easy understanding.
<Scheme A>
In the example of FIG. 8(b), the irradiation diameter of the laser beans is set irrespective of the size of the unit attention region 102, that is, irrespective of the step width of the laser beans irradiation position, and the laser beam irradiation position is moved with a step width corresponding to the size of the unit attention region 102 while each unit attention regions 102 is irradiated with laser beans. In this case, in the analysis target region 101, a large portion of the analysis target region 101 is not irradiated with laser beam, which produces a non-ionized region 104. Consequently, use efficiency of the sample is low and the amount of generated ions is small, so that the high-sensitivity analysis cannot be performed. Additionally, the substance existing only in the region that is not irradiated with the laser beam is not reflected at all in the mass spectrometry result, so that there is a risk that an important substance is overlooked.
<Scheme B>
For example, in the imaging mass spectrometer disclosed in Patent Document 1, the irradiation diameter of the laser beam with which the sample is irradiated can be adjusted. In the example of FIG. 8(c), the irradiation diameter of the laser beam is adjusted according to the size of the unit attention region 102, that is, the step width of the laser beam irradiation position. Specifically the irradiation diameter of the laser beam is adjusted such that the size of the unit attention region 102 is substantially matched with the irradiation diameter of the laser beam, and the laser beam irradiation position is moved with the step width corresponding to the size of the unit attention region 102 while each unit attention region 102 is irradiated with the laser beam. Even in this case, a non-ionized region 104 inevitably remains at the four corners of each unit attention region 102.
<Scheme C>
In the example of FIG. 8(d), although the irradiation diameter of the laser beam is equal to that of the example in FIG. 8(b), the step width of the laser beam irradiation position is narrowed so as to match with the irradiation diameter of the laser beam, and a plurality of analyses are performed on different minute regions in one unit attention region 102 (see Non-Patent Document 2). The mass spectrometry results obtained in the different minute regions of one unit attention region 102 are integrated or averaged to calculate the mass spectrometry result for the unit attention region 102. In this case, unlike the scheme B, it is unnecessary to adjust the irradiation diameter of the laser beam, so that a mechanism that changes the irradiation diameter of the laser beam is not required. At the same time, because the number of analyses and the number of moving times of the irradiation position of the laser beam are increased as compared with the scheme B, there is a disadvantage that a total analysis time is prolonged. Even in this case, a non-ionized region 104 inevitably remains at the four corners of the rectangular region circumscribing substantially circular laser beam irradiation region.
<Scheme D>
In the example of FIG. 8(e), similarly to the scheme B, the irradiation diameter of the laser beam is increased and the laser beam irradiation position is moved with a predetermined step width smaller than the size of the unit attention region 102 (in this example, a step width of about a half of the size in the X-axis direction or the Y-axis direction of the unit attention region 102) (see Non-Patent Document 3). In the above schemes A to C, the laser beams with which the different laser beam irradiation positions are irradiated do not overlap each other. However, in the scheme D, the laser beams with which the laser beam irradiation positions adjacent to each other are irradiated overlap each other. As a result, the non-ionized region 104 is avoided except a periphery of the analysis target region 101. Thus, only a small part of the non-ionized region 104 remains along the periphery of the analysis target region 101, and the use efficiency of the sample is very close to 100%.
However, in the scheme D, because a laser beam irradiation region extends over adjacent unit attention regions 102, the association between the position of the unit attention region 102 and the mass spectrometry result becomes complicated. Additionally, the scheme D has the following problems.
The amount of substance existing in each laser beam irradiation region is finite. If, a mass spectrometry is performed by irradiating a certain region with a laser beam and then the same region is irradiated with another laser beam, the amount of ions generated considerably decreases. For this reason, when the laser beam irradiation regions partially overlap each other, the amount of ions generated corresponding to the subsequent laser beam irradiation becomes small.
FIG. 9 is a diagram illustrating an example of a relationship between every laser beam irradiation position and the shape of the region where the sufficient amount of ions is obtained. The laser beam irradiation position is moved from the unit attention region 102 located at the left uppermost position in the analysis target region 101 along the X-axis direction with the step width (the bold-line arrow in FIG. 9), the scanning returns to a left end when reaching the right end of the analysis target region 101, and the laser beam irradiation position is moved in the Y-axis direction with the step width. In this way, the scanning is performed such that the laser beam irradiation position is finally moved to the lower right end of the analysis target region 101. In this case, suppose no ion is generated in the region already irradiated with the laser beam, the area in which the ionization can be performed with sufficiently high efficiency in each laser beam irradiation region is not the same as illustrated in FIG. 9. Consequently, the sensitivity is relatively low at the central portion of the analysis target region 101 as compared with the peripheral portion. That is, even if a certain substance is uniformly distributed in the analysis target region 101, a nonuniformity occurs in that the signal intensity of the ions of the substance is higher in the periphery as compared with the central portion. The nonuniformity varies depending on the scanning direction or scanning order of the laser beam irradiation position.